Molecules, compositions and methods for modulation of SIRT6

ABSTRACT

A SIRT6 activating molecule as shown in Tables 1-3 or a compound according to a pharmacophore as described herein.

FIELD OF THE INVENTION

The present invention relates to novel molecules and compositionscontaining same for modulation of SIRT6, and methods of preparation andadministration thereof.

BACKGROUND OF THE INVENTION

Sir2 family of enzymes, namely sirtuins, consists of seven mammalianenzymes (SIRT1 to SIRT7) that share a conserved core catalytic domain,but differ in tissue specificity, subcellular localization, enzymaticactivity and targets. The sirtuins are NAD+-dependent enzymes thatregulate a large number of diverse cellular pathways. Among thesirtuins, SIRT6, a chromatin-associated enzyme with deacetylase andlong-chain deacylase activities, is considered to have a leading role inregulating genomic stability, cellular metabolism, stress response, andaging. SIRT6-deficient mice display shortened lifespan and acutedegenerative and metabolic defects similar to premature agingpathologies, including loss of subcutaneous fat, lordokyphosis, colitisand severe lymphopenia. Additionally, transgenic mice overexpressingSIRT6 have a significantly longer life span than wild-type control mice,further supporting that SIRT6 is central in regulating aging processes.Furthermore, SIRT6 regulates glucose homeostasis and fat metabolism andsuppresses obesity, fatty liver, glucose intolerance, inflammation,cardiac hypertrophy and cellular senescence.

SUMMARY OF THE INVENTION

Accumulating data indicate that SIRT6 is the sirtuin with specialrelevance for regulating aging processes and metabolism. SIRT6 isinvolved in several DNA repair pathways and associates specifically withtelomeres. Inactivation of SIRT6 leads to hypersensitivity to DNAdamage, loss of telomere protection and genomic instability. Thus, SIRT6protects tissues from telomere dysfunction, promotes genome stabilityand resistance to DNA damage and oxidative stress, the principal defectsassociated with age-related diseases and cancer. As genomic instabilityand altered metabolism are hallmarks of many cancers, SIRT6 was shown toact as tumor suppressor also by controlling cancer metabolism(inhibition of Warburg effect). Moreover, SIRT6 was found to be a majorregulator of glucose homeostasis and fat metabolism. SIRT6 is a criticalregulator of cholesterol homeostasis and involved in regulation oflipogenesis by repression of lipogenic transcription factors, SREBP1 andSREBP2. SIRT6 transgenic mice fed a high fat diet are protected againstfat accumulation, elevated triglyceride and LDL cholesterol levels andimpaired glucose tolerance. The involvement of SIRT6 in fat metabolismis further supported by the finding that liver specific SIRT6 deletionresults in liver steatosis. In addition, overexpression of SIRT6improves aspects of age-associated metabolic decline such as glucoseintolerance. On the whole, SIRT6 is a key regulator of metabolichomeostasis, the imbalance of which eventually accelerates aging andsenescence.

SIRT1 was originally thought to be the most important sirtuin involvedin aging and other disease processes, a role which is now believed toalso be held by SIRT6. However, it has recently been found that SIRT1and SIRT6 may in fact have different roles in various disease processes,for example with regard to metabolic diseases. An article by Masri et al(Cell, 2014, vol 158, pages 659-672) illustrates a difference betweenSIRT1 and SIRT6 in controlling cellular metabolism. SIRT1 and SIRT6,manage important liver processes (lipid storage and energy usage)separately and distinctly from each other. In an interview, the authorsclaimed(http://news.uci.edu/press-releases/strict-genomic-partitioning-by-biological-clock-separates-key-metabolic-functions/)that these findings may contribute to the design of pharmacologicalstrategies targeting SIRT1- or SIRT6-specific metabolic functions andpathologies. Thus, SIRT6 specific modulation would be of clearimportance for treating various diseases.

The background art does not teach or suggest effective molecules,compositions containing same, or methods of preparation and use thereof,for the modulation of SIRT6.

According to at least some embodiments, there are provided effectivemolecules, compositions containing same, or methods of preparation anduse thereof, which are believed to be useful for the modulation of SIRT6(without wishing to be limited by single hypothesis in terms of otheradditional or different activities that these molecules may have). Thesemolecules are presented below in Tables 1-3, in Example 1, whichdescribes the assay used to test these molecules for SIRT6 activity. Themolecules in Tables 1-3 all showed the ability to activate SIRT6.

According to at least some embodiments, the SIRT6 modulators mayoptionally be used to treat obesity or an obesity-related disease, or afat-related metabolic disorder.

“Obesity” refers to a condition in which a subject has a body mass indexof greater than or equal to 30. “Over-weight” refers to a condition inwhich a subject has a body mass index of greater or equal to 25.0. Thebody mass index and other definitions are according to the “NIH ClinicalGuidelines on the Identification and Evaluation, and Treatment ofOverweight and Obesity in Adults” (1998). In particular, obesity canlead to type II diabetes in successive phases. Clinically, these phasescan be characterized as normal glucose tolerance, impaired glucosetolerance, hyperinsulinemic diabetes, and hypoinsulinemic diabetes. Sucha progressive impairment of glucose storage correlates with a rise inbasal glycemia.

“Obesity-related disease” and “Fat-related metabolic disorder” include,but are not limited to, anorexia nervosa, wasting, AIDS-related weightloss, bulimia, cachexia, lipid disorders including hyperlipidemia andhyperuricemia, insulin resistance, noninsulin dependent diabetesmellitus (NIDDM, or Type II diabetes), insulin dependent diabetesmellitus (IDDM or Type I diabetes), diabetes-related complicationsincluding microangiopathic lesions, ocular lesions, retinopathy,neuropathy, and renal lesions, cardiovascular disease (including cardiacinsufficiency, coronary insufficiency, and high blood pressure),atherosclerosis, atheromatous disease, stroke, hypertension, Syndrome X,gallbladder disease, osteoarthritis, sleep apnea, forms of cancer suchas uterine, breast, colorectal, kidney, and gallbladder, highcholesterol levels, complications of pregnancy, menstrualirregularities, hirsutism, muscular dystrophy, infertility, aweight-related disorder (characterized by a subject being over or underweight, e.g., being within the top or bottom 25th percentile of bodymass index) and increased surgical risk. In preferred embodiments, atreated or diagnosed subject is a mammal, preferably a human.

Fat-related metabolic disorders include disorders in which (i) increasedfat storage, reduced fat mobilization, and/or reduced fat burning isdesired, and (ii) other disorders in which reduced fat storage,increased fat mobilization and/or increased fat burning is desired.Examples of the first category of disorders include, e.g., anorexianervosa, wasting, AIDS-related weight loss, bulimia, cachexia. Examplesof the latter category include, e.g., obesity, cardiovascular disease,osteoarthritis. The classification of other disorders (e.g.,infertility, increased surgical risk, pregnancy complications) maydepend on the weight of the subject, e.g., whether the subject is over-or underweight. Overweight subjects can be treated, e.g., with an agentthat increases SIRT6 activity, and underweight subject can be treated,e.g., with an agent that decreases SIRT6 activity.

Optionally, the above diseases may also be treatable by SIRT6 modulatorsin cases where the subject is not obese according to the definition ofBMI (body mass index), which requires a BMI of at least 30 for obesity.Instead, the above diseases may also optionally be treatable by SIRT6modulators in subjects in which body tissue composition, such as forexample body fat percentage, results in one of the above diseases.Furthermore, SIRT6 modulators may also optionally be able to treatsubjects suffering from sub-optimal body tissue composition, such as forexample higher than desired body fat percentage. While the definition ofhigher than desired body fat percentage may vary, it may for examplerelate to a percentage in any integral amount starting at least at 20%,at least at 25%, at least at 30%, at least at 40%, at least at 45% andany amount in between.

SIRT6 modulators may also optionally be used to treat subjects sufferingfrom sub-optimal body fat distribution, such as for example excessabdominal fat, which has been shown to lead to a higher risk of variousdiseases, including without limitation the above diseases. SIRT6modulators may optionally be used to treat body fat distributiondirectly and/or one of the above diseases associated with obesity orfat-related metabolic disorders.

Sirt6 modulators may also optionally be used to treat metabolic diseasesthat are not necessarily related to fat or to obesity, and/or in whichthe subject is not obese or even overweight. For such diseases,metabolic disorders are defined as conditions, diseases, and disordersassociated with glucose and/or lipid metabolism dysregulation, includingbut not limited to: type 2 diabetes, prediabetes, glucose intolerance,insulin insensitivity, hyperglycemia, hypoglycemia, gestationaldiabetes, drug-induced diabetes; overweight, obesity, high percent bodyfat and/or body composition and/or body fat location, dyslipidemiadefined as abnormal levels of blood lipids such as cholesterol,triglycerides and free fatty acids; and non-alcoholic fatty liverdisease (NAFLD) ranging from steatosis to steatohepatitis (NASH),advanced fibrosis and cirrhosis.

According to at least some embodiments, the SIRT6 modulators mayoptionally be used to treat diseases associated with aging andsenescence, including but not limited to cardiovascular disease,neurodegenerative diseases, premature aging syndromes and aging.Optionally and preferably, these diseases are selected from the groupdescribed below (described according to various physiological systemsfor the sake of clarity only and without any intention of beinglimiting):

SKIN: Loss of subcutaneous fat, Decrease in Collagen elasticity,Xerosis, Senile Purpura, Pruritis, Pressure ulcers, Venous and Arterialulcers, Skin cancer, Aging-related skin lesions such as Skin tags(Acrochordon) and Keratosis.

SKELETAL SYSTEM: Osteoporosis, Arthritis, Osteomalacia, Paget's disease

MUSCLES: Muscle wasting and/or atrophy, Muscle cramps, MyastheniaGravis, Polymyalgia Rheumatica, Bursitis

NERVOUS SYSTEM: Tremor, Parkinson's disease, Tardive Dyskinesia, SleepDisorders, Brain tumors, Delirium, Various forms of Dementia includingAlzheimer's disease.

SENSORY SYSTEM: Cataract, Glaucoma, Diabetic Retinopathy, Age-RelatedMacular Degeneration (AMD), Hearing loss, Tinnitus, Otosclerosis,Meniere's disease, Olfactory distortion.

CARDIOVASCULAR SYSTEM: Arteriosclerosis (lessened elasticity of arterywalls), Atherosclerosis (fatty deposits on inner walls of arteries),Hypertension, Postural Hypotension, Acute Coronary Syndrome, AnginaPectoris, Myocardial Infraction, Congestive Heart Failure (CHF), HeartValve disease, Cardiac Arrhythmias and Conduction disorders, TransientIschemic Attack (mini-stroke), Cerebrovascular accident (Stroke),Aneurysm, Arterial Occlusion, Age-dependent increase of thromboembolicevents

RESPIRATORY SYSTEM: Chronic Obstructive Pulmonary disease (COPD),Chronic Bronchitis, Emphysema, Pulmonary Tuberculosis (TB), Pneumonia,Lung cancer.

GASTROINTESTINAL SYSTEM: Age-Related Disorders of the Mouth, Esophagus,Stomach, Small Intestine, Large Intestine, Pancreas, Liver andGallbladder including Xerostomia (dry mouth), Dysphagia (difficultyswallowing), Periodontal disease, Oral cancer, Gastroesophageal Refluxdisease, Hiatal Hernia, Cancer of the Esophagus, Gastritis (inflammationof the stomach), Gastric Ulcer, Cancer of the Stomach, Cancer of theColon and Rectum, Hemorrhoids, Cancer of the Pancreas, Cirrhosis,Gallstones (Cholelithiasis).

ENDOCRINE SYSTEM: Pancreatitis, Pancreatic cancer

URINARY SYSTEM: Urinary Tract Infections (UTIs), Cystitis (lower urinarytract infection), Pyelonephritis (upper urinary tract infection), AcuteGlomerulonephritis, Benign Prostatic Hyperplasia (BPH), Nephrolithiasis(kidney stone disease), Cancer of the Bladder, Urinary Incontinence,Renal Failure.

REPRODUCTIVE SYSTEM: 1. Female: Atrophic Vaginitis, Pelvic OrganProlapse, Cancer of the cervix, uterus, ovary or breast. 2. Male: Cancerof the Prostate

According to at least some embodiments, the SIRT6 modulators mayoptionally be used to treat cancer, optionally selected from the groupconsisting of but not limited to breast cancer, cervical cancer, ovarycancer, endometrial cancer, melanoma, uveal melanoma, bladder cancer,lung cancer, pancreatic cancer, colorectal cancer, prostate cancer,leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia,B-cell lymphoma, Burkitt's lymphoma, multiple myeloma, Non-Hodgkin'slymphoma, myeloid leukemia, acute myelogenous leukemia (AML), chronicmyelogenous leukemia, thyroid cancer, thyroid follicular cancer,myelodysplastic syndrome (MDS), fibrosarcomas and rhabdomyosarcomas,teratocarcinoma, neuroblastoma, glioma, glioblastoma, benign tumor ofthe skin, keratoacanthomas, renal cancer, anaplastic large-celllymphoma, esophageal cancer, follicular dendritic cell carcinoma,seminal vesicle tumor, epidermal carcinoma, spleen cancer, bladdercancer, head and neck cancer, stomach cancer, liver cancer, bone cancer,brain cancer, cancer of the retina, biliary cancer, small bowel cancer,salivary gland cancer, cancer of uterus, cancer of testicles, cancer ofconnective tissue, myelodysplasia, Waldenström's macroglobinaemia,nasopharyngeal, neuroendocrine cancer, mesothelioma, angiosarcoma,Kaposi's sarcoma, carcinoid, fallopian tube cancer, peritoneal cancer,papillary serous mullerian cancer, malignant ascites, gastrointestinalstromal tumor (GIST), Li-Fraumeni syndrome, and Von Hippel-Lindausyndrome (VHL).

According to at least some embodiments, the SIRT6 modulators mayoptionally be used to treat cancer, optionally selected from the groupconsisting of but not limited to breast cancer, cervical cancer, ovarycancer, endometrial cancer, melanoma, uveal melanoma, bladder cancer,lung cancer, pancreatic cancer, colorectal cancer, prostate cancer,leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia,B-cell lymphoma, Burkitt's lymphoma, multiple myeloma, Non-Hodgkin'slymphoma, myeloid leukemia, acute myelogenous leukemia (AML), chronicmyelogenous leukemia, thyroid cancer, thyroid follicular cancer,myelodysplastic syndrome (MDS), fibrosarcomas and rhabdomyosarcomas,teratocarcinoma, neuroblastoma, glioma, glioblastoma, benign tumor ofthe skin, keratoacanthomas, renal cancer, anaplastic large-celllymphoma, esophageal cancer, follicular dendritic cell carcinoma,seminal vesicle tumor, epidermal carcinoma, spleen cancer, bladdercancer, head and neck cancer, stomach cancer, liver cancer, bone cancer,brain cancer, cancer of the retina, biliary cancer, small bowel cancer,salivary gland cancer, cancer of uterus, cancer of testicles, cancer ofconnective tissue, myelodysplasia, Waldenstrom's macroglobinaemia,nasopharyngeal, neuroendocrine cancer, mesothelioma, angiosarcoma,Kaposi's sarcoma, carcinoid, fallopian tube cancer, peritoneal cancer,papillary serous mullerian cancer, malignant ascites, gastrointestinalstromal tumor (GIST), Li-Fraumeni syndrome, and Von Hippel-Lindausyndrome (VHL).

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin order to provide what is believed to be the most useful and readilyunderstood description of the principles and conceptual aspects of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for a fundamentalunderstanding of the invention, the description taken with the drawingsmaking apparent to those skilled in the art how the several forms of theinvention may be embodied in practice.

FIG. 1 shows the area under the peak corresponding to the amount ofdeacylated product after incubation with the compounds, in comparison tothe DMSO control;

FIG. 2 shows the effect of compounds on SIRT6 deacetylation activity;

FIG. 3 shows deacetylation of H3K9 on chromatin histones by SIRT6, inthe presence or absence of compound D715-0284; and

FIGS. 4A-4E show exemplary pharmacophores for E677-1326, E677-0945 andD715-0284.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1—Description of SIRT6Activating Molecules and Demonstration of their Activity

Tables 1-3 below show some exemplary SIRT6 activating molecules, as wellas their activity levels in a particular assay, as described below.Without wishing to be limited in any way, it is noted that the moleculesin Table 1 showed greater activity in this particular assay.

Expression and Purification of SIRT6 Protein

Human recombinant SIRT6 (sequence identifier: CAG33481.1; GI:4814651)was expressed in large scale in Escherichia coli strain BL21 as asix-histidine-tagged protein, after transformation of the expressionplasmid pET28-SIRT6. The SIRT6 enzyme was purified from the bacteriaculture broth in a two-step strategy. The first purification step wascarried out by cation exchange chromatography on a HiTrap CM-Sepharosecolumn (GE Healthcare) using an FPLC system. The second purificationstep was performed by immobilized metal ion affinity chromatography(IMAC) using a cobalt affinity column (Clontech).

Method of Assay

The enzymatic activity assay is carried out in 96 or 384-wellmicroplates using the purified recombinant SIRT6 and afluorophore-labeled peptide substrate: 7-amino-4-methylcoumarin (AMC)that is quenched through conjugation to the C-terminal end of aLysin-myristoylated histone H3 peptide. The activity assay procedurerequires two steps, both performed in the same microplate. In the firststep, the substrate is incubated with the purified SIRT6 protein alongwith the co-substrate nicotinamide adenine dinucleotide (NAD+). Uponde-myristoylation, the liberated ε-amino group of the lysine becomes atrypsin substrate. Treatment with trypsin in the second step releasesthe fluorophore resulting in an increase in fluorescence. Fluorescenceintensity is measured using a fluorescent plate reader (excitation andemission at 370 nm and 450 nm, respectively). The fluorescence signal isdirectly proportional to SIRT6 enzymatic activity.

High Throughput Screening

In the primary screening, compounds from a library (ChemDiv Inc.) weretested at a single concentration of 20 micromolar for their potential tomodulate SIRT6 enzymatic activity. The assay was carried out in 384-wellblack well plates and all liquid handling was done using a roboticliquid handler. Purified recombinant SIRT6 (1 micromolar) waspre-incubated with each compound (or DMSO in control wells) for 15minutes at room temperature. The first stage of the assay was initiatedby adding a ‘substrate solution’ containing the myristoylated histone H3peptide and NAD+(for final concentrations of 25 micromolar and 20micromolar, respectively). After one hour incubation at 37° C., aTrypsin solution (6 mg/mL) was added for 30 minutes at room temperature.Fluorescence intensity was measured using a fluorescent plate reader(Tecan) with excitation at 370 nm and emission at 450 nm. The effect ofeach tested compound was evaluated by the change in the fluorescenceintensity compared to control wells. The compounds that demonstratedhigher fluorescence intensity in comparison with control wells wereconsidered as primary “hits”. ‘Activator’ activity is defined as thepercentage of signal increase relative to the average signal in controlwells. To validate the primary screening results, each of the primary“hits” was re-tested, first at concentration of 20 micromolar induplicates and later at a serial dilution ranging from 100 to 0.2micromolar in triplicates.

Results of Assay—

The results of the assay are given below in Tables 1-3. For each table,column 1 gives an internal identifier; column 2 gives the CAS number forthe molecule while column 3 shows its ChemDiv ID; column 4 gives themolecular structure; and column 5 shows percent activity of the moleculein the above assay, at two concentrations: 20 micromolar (left) and 100micromolar (right). It should be noted that molecule “c” (third row ofTable 1) is a specific stereoisomer as the other stereoisomer was notshown to be active in this assay (data on the other stereoisomer notshown, which has the CAS number 768382-41-8, as no specific CAS numberhas been assigned to the current stereoisomer in the table; this CASnumber only to represents the other stereoisomer that is not shown).

TABLE 1 % activity ChemDiv Max. conc. # CAS number ID Structure 20 uM100 uM A 931369-52-7 E677-0945

263 255 B 931312-00-4 E677-1326

287 252 C stereoisomer of molecule 768382-41-8 D715-0284

70 195

TABLE 2 ChemDiv % activity # CAS number ID Structure 20 uM 100 uM 1332173-46-3 2803-0068

88 428 2 351469-45-9 3389-1644

29 46 3 443677-66-5 4285-2380

25 33 4 328286-54-0 4477-0762

29 56 5 375359-94-7 4477-2607

39 54 6 353516-56-0 4896-3248

30 28 7 797029-49-3 6843-3168

118 162 8 339156-78-4 8001-8196

25 25 9 99254-31-6 8008-0525

22 48 10 300588-35-6 8008-8279

25 27 11 70598-96-8 8010-0818

41 99 12 442642-14-0 8012-5593

36 59 13 515875-10-2 8013-3307

24 86 14 24386-17-2 8014-9229

28 48 15 290299-89-7 8018-3339

37 75 16 128294-19-9 8018-4551

40 197 17 879058-43-2 8018-6508

21 18 18 879765-85-2 8020-1200

53 94 19 1005160-87-1 C142-0098

37 61 20 1031990-46-1 C201-1744

78 22 21 901014-49-1 C381-0232

38 36 22 902559-22-2 C535-0969

32 29 23 1017523-49-7 D506-0127

55 65 24 1293946-11-8 D549-0050

77 47 25 1358629-50-1 D577-0290

26 22 26 1474056-79-5 D715-0292

21 56 27 950279-39-7 E146-0990

59 137 28 938025-58-2 F025-0094

134 246 29 938026-06-3 F025-0098

82 226 30 938025-40-2 F025-0102

149 111 31 932969-43-2 F498-0132

29 16 32 1040683-42-8 G744-0071

101 73 33 1040572-56-4 G744-0080

53 221 34 1206085-08-6 G755-0539

34 87 35 933226-39-2 G891-0058

154 324 36 1446199-09-2 H024-0026

45 56 37 422272-66-0 K232-1782

42 35 38 899707-19-8 K906-5001

52 52 39 434925-30-1 K937-0320

32 54 40 1115352-35-6 L036-0162

62 92 41 1115421-77-6 L036-0163

40 110 42 1115317-33-3 L036-0174

61 87 43 1115317-58-2 L036-2289

96 119 44 1115370-79-0 L103-0652

34 42 45 1115370-86-9 L103-0696

91 159 46 1112327-81-7 M049-1557

51 56 47 1340850-02-3 M715-0111

63 50 48 1340765-63-0 M715-0112

40 54 49 1341007-09-7 M715-0156

93 75 50 1358902-84-7 M723-0069

30 51 51 1243005-49-3 M723-1403

23 87 52 1358277-15-2 M723-1459

69 142 53 1091996-67-6 M830-6517

36 22 54 1358236-69-7 M830-6572

31 11 55 1358351-15-1 P613-0273

31 37 56 1357727-05-9 P933-1294

30 53 57 1340696-21-0 P950-0537

37 45 58 1775562-29-2 S051-0094

38 47 59 1203182-84-6 V004-5575

89 175 60 1314203-44-5 V030-4109

52 255 61 1314211-30-7 V030-4128

50 210 62 1173666-66-4 Y040-3625

28 113

Table 3 shows an additional two structures with data at the 20micromolar concentration only

% activity (20 ChemDiv micromolar # CAS number ID Structure only) 631357759-28-4 M535-1335

38% 64 902840-30-6 E205-0117

85%

Example 2—Compositions and Methods of Administration for SIRT6Activating Molecules

Pharmaceutical Compositions

As used herein, the term “excipient” means the substances used toformulate active pharmaceutical ingredients (API) into pharmaceuticalformulations; in a preferred embodiment, an excipient does not lower orinterfere with the primary therapeutic effect of the API. Preferably, anexcipient is therapeutically inert. The term “excipient” encompassescarriers, diluents, vehicles, solubilizers, stabilizers, bulking agents,acidic or basic pH-adjusting agents and preservatives. Excipients canalso be those substances present in a formulation as an indirect orunintended result of the manufacturing process.

The term “sublingual administration” refers to the mode ofadministration of a medicament to the tissue under the tongue.

“Treating” or “treatment” of a disease includes: (1) preventing thedisease, i.e., causing the clinical symptoms of the disease not todevelop in a mammal that may be exposed to or predisposed to the diseasebut does not yet experience or display symptoms of the disease, (2)inhibiting the disease, i.e., arresting or reducing the development ofthe disease or its clinical symptoms, or (3) relieving the disease,i.e., causing regression of the disease or its clinical symptoms.

According to some embodiments, the composition may be administered in aform of a sublingual spray for quick systemic distribution, rapidtherapeutic activity and ease of use.

In another embodiment, the sublingual spray formulations of the presentinvention can take various forms including, but not limited to, aqueoussolutions, non-aqueous solutions and combinations thereof. Aqueoussolutions include, for example, aqueous gels, aqueous suspensions,aqueous liposomal dispersions, aqueous emulsions, aqueous microemulsionsand combinations thereof. Non-aqueous solutions include, for example,non-aqueous gels, non-aqueous suspensions, non-aqueous liposomaldispersions, non-aqueous emulsions, non-aqueous microemulsions andcombinations thereof.

In another embodiment, the pH of the compositions may be maintained fromabout 3.0 to about 10.0. Compositions having a pH of less than about 3.0or greater than about 10.0 can increase the risk of irritating themucosal membranes in the sublingual region of a recipient. In anotherembodiment, the pH of the compositions is maintained from about 3.0 toabout 7.0.

According to some embodiments, preservatives may be added to the presentcompositions. Suitable preservatives that can be used with the presentcompositions include benzyl alcohol, parabens, thimerosal, chlorobutanoland benzalkonium chloride and preferably benzalkonium chloride is used.In another embodiment, the preservative are present in a composition ina concentration of up to about 2% by weight.

In another embodiment, the compositions of the invention can beformulated so as to provide quick, sustained or delayed release of theactive ingredient after administration to the patient by employingprocedures known in the art. In another embodiment, the formulation mayalso comprise a muco-adherent to increase the residence time on themucosa; including chitosan, polyvinyl pyrrolidone, or gelatin.

In another embodiment, the formulation may further comprise amoisturizing agent, such as propylene glycol, or polyethylene glycol.The formulation may further comprise an antioxidant, such as butylatedhydroxyltoluene, ascorbic acid, alkyl gallates, or tocopherols. Theformulation may further comprise an ionic or nonionic surfactant, suchas sodium lauryl sulfate, or sorbitan esters.

In another embodiment, the formulation may further comprise aco-solvent. In another embodiment, the organic solvent is an alcohol. Inanother embodiment, the alcohol may comprise, but is not limited to,ethanol, propylene glycol, glycerol, polyethylene glycol and mixturesthereof. More preferably, the alcohol is ethanol. In another embodiment,the organic solvent is present in an amount of 0-90% w/w.

According to some demonstrative embodiments, the present invention mayprovide for compositions as described above which may be administered tothe sublingual tissue under the tongue to a mammal to treatFibromyalgia. In another embodiment, the formulation is administered asa spray. In another embodiment, the spray is administered directly tothe sublingual mucosa, i.e., the formulation is sprayed directly ontothe tissue under the patient's tongue.

In some demonstrative embodiments, the composition of the presentinvention may be administered in any suitable form for sublingualadministration, including but not limited to, sublingual tablets, forexample, any tablets which may easily melt in the mouth, dissolverapidly and with little or no residue; multi-purpose tablets, e.g,soluble tablets for either oral or sublingual (or buccal)administration; sublingual drops, e.g., a concentrated solution and/or aliquid form of the composition of the present invention adapted to bedropped under the tongue; sublingual spray; lozenges, e.g, which mayeffect a metered and patient-controlled-rate combination of sublingual,buccal, and oral administration; effervescent buccal or sublingualtablets; bioadhesive sublingual tablets; dissolving films

According to some embodiments, the preferred administration form may bea sublingual spray.

According to some demonstrative embodiments, the composition of thepresent invention may be formulated in the form of a dissolvingsublingual tablet.

According to some embodiments, the tablet may be prepared using acompression molding process and the tablet may exhibit rapiddisintegration and dissolution, which is usually within 5-10 seconds.

In some demonstrative embodiments, the tablet (whether for sublingual ororal administration) may be prepared using direct compression. Accordingto some embodiments, the direct compression method may employingredients that can be mixed well and do not require furthergranulation steps prior to lubrication and compression.

The directly compressible tablet formulation may contain directlycompressible soluble excipients, a super disintegrant, and lubricant.According to some embodiments, the formulation may also includemicrocrystalline cellulose, dry binder, buffers, surface-active agents,sweeteners, and flavors. Sugar-based excipients may also be used in theformulation as bulking agents because of their high aqueous solubility,sweetness, pleasant feeling in the mouth, and good taste-masking.

According to some demonstrative embodiments, the composition may beadministered in a tablet or capsule form which may include a substratecomprising the active ingredient covered with at least one entericcoating.

According to some embodiments, the enteric coating may provideprotection upon exposure of the composition to the acidic conditions ofthe stomach, and may dissolve when the composition is further digestedthroughout the Gastrointestinal (GI) tract, e.g., when reaching thesmall intestine.

Non-limiting examples of enteric coatings, may include suitable polymerssuch as cellulose acetate phthalate (CAP); hydroxypropyl methylcellulosephthalate (HPMCP); polyvinyl acetate phthalate; cellulose acetatetrimellitate; poly((methacrylic acid, methyl methacrylate)1:1) (EudragitL100™), poly((methacrylic acid, ethyl acrylate)1:1) (Eudragit L30D-55)or Eudragit L100-55™, (poly(methacrylic acid, methyl methacrylate)1:2)Eudragit™ S hydroxypropyl methylcellulose acetate succinate (HPMCAS),sodium alginate, and alginic acid or mixtures thereof.

According to some demonstrative embodiments, the present inventionprovides for granular compositions.

According to some embodiments, a composition containing the activeingredient may mixed and granulated with a suitable carrier material toform a core part of particles to be coated.

In some embodiments, the granulation process may employ a suitablegranulator, or alternatively a fluidized bed. The drying process maycomprise lyophilization. The granules according to the invention mayhave a wide range of dimensions. A non-limiting example of a granuleaccording to the invention is an essentially spherical particle having amean diameter of about from 0.1 to about 1000 microns.

According to some demonstrative embodiments, the enteric coatingdescribed herein may comprise one or more pH-sensitive coatings,according to conventional procedures in order to delay the release ofthe active ingredient. Suitable pH-sensitive polymers include thosewhich are relatively insoluble and impermeable at the pH of the stomach,but which are more soluble or disintegrate or permeable at the pH of thesmall intestine and colon. Such pH-sensitive polymers includepolyacrylamides, phthalate derivatives such as acid phthalate ofcarbohydrates, amylose acetate phthalate, cellulose acetate phthalate(CAP), other cellulose ester phthalates, cellulose ether phthalates,hydroxypropylcellulose phthalate (HPCP), hydroxypropylethyl cellulosephthalate (HPECP), hydroxylpro-plymethylcellulose phthalate (HPMCP),HPMCAS, methylcellulose phthalate (MCP), polyvinyl acetate phthalate(PVAcP), polyvinyl acetate hydrogen phthalate, sodium CAP, starch acidphthalate, cellulose acetate trimellitate (CAT), styrene-maleic aciddibutyl phthalate copolymer, styrene-maleic acid/polyvinylacetatephthalate copolymer, styrene and maleic acid copolymers, polyacrylicacid derivatives such as acrylic acid and acrylic ester copolymers,polymethacrylic acid and esters thereof, polyacrylic and methacrylicacid copolymers, shellac, and vinyl acetate and crotonic acidcopolymers. Preferred pH-sensitive polymers include shellac, phthalatederivatives, CAT, HPMCAS, polyacrylic acid derivatives, particularlycopolymers comprising acrylic acid and at least one acrylic acid ester,polymethyl methacrylate blended with acrylic acid and acrylic estercopolymers, and vinyl acetate, crotonic acid copolymers, alginic acidand alginates such as ammonia alginate, sodium, potassium, magnesium orcalcium alginate. A particularly preferred group of pH-sensitivepolymers includes CAP, PVAcP, HPMCP, HPMCAS, anionic acrylic copolymersof methacrylic acid and methylmethacrylate, and osmopolymers comprisingacrylic acid and at least one acrylic acid ester. Cellulose acetatephthalate may be applied as an enteric coating to the encapsulatedpro-biotic compositions of the invention to provide delayed release ofthe active ingredient until the dosage form has exited the stomach. TheCAP coating solution may also contain one or more plasticizers, such asdiethyl phthalate, polyethyleneglycol-400, triacetin, triacetin citrate,propylene glycol, and others as known in the art. Preferred plasticizersare diethyl phthalate and triacetin. The CAP coating formulation mayalso contain one or more emulsifiers, such as polysorbate-80. Anionicacrylic copolymers of methacrylic acid and methylmethacrylate are alsoparticularly useful enteric coating materials for delaying the releaseof the active ingredient until they have moved to a position in the GItract which is distal to the stomach. Copolymers of this type mayinclude anionic copolymers of methacrylic acid and methylmethacrylate.Preferred non-pH-sensitive aqueous insoluble polymers may includecellulose esters, cellulose ethers, polyacrylates, polyamides,polyesters, and vinyl polymers. Preferred non-pH-sensitiveaqueous-soluble polymers include hydroxyalkyl-substituted cellulosicssuch as HPC, HEC and HPMC, PVA, PEG, PEO, PEG/PPG copolymers, andaqueous-soluble polyamides, polysaccharides, and polyacrylates.

Various additives may be included in such coatings, includingemulsifiers, plasticizers, surfactants, fillers and buffers. Finally,the polymeric coating may be described as being “quasi-enteric” in thesense that it remains substantially intact for a significant period oftime (e.g., greater than an hour) after the dosage form exits thestomach, thereafter becoming sufficiently permeable to permit gradualrelease of the active ingredient and diffusion through the coating.

Optionally and preferably, the substrate is an active core forcontaining the active ingredient, such as, for example, a pellet, a beador a tablet.

Optionally and preferably, the active core is a tablet formed bycompression.

In some demonstrative embodiments, the substrate may further comprise afiller, such as, for example, one or more of microcrystalline cellulose,sodium carboxymethycellulose, ethylcellulose, cellulose acetate, starch,lactose, glucose, fructose, sucrose, dicalcium phosphate, sorbitol,mannitol, mantitol, lactitol, xylitol, isomalt, erythritol, andhydrogenated starch hydrolysates, or a mixture thereof.

In some demonstrative embodiments, the substrate may further comprise adisintegrant, such as, for example, one or more of low-substitutedcarboxymethyl cellulose sodium, cross-linked polyvinyl pyrrolidone,sodium starch glycolate, cross-linked sodium carboxymethyl cellulose,pregelatinized starch, microcrystalline starch, water insoluble starch,calcium carboxymethyl cellulose, and low substituted hydroxypropylcellulose magnesium aluminum silicate, or a mixture thereof.

Example 3—Additional Validation of Three Molecules

The activating effect of three compounds (ChemDiv ID E677-1326,E677-0945 and D715-0284) for SIRT6 was validated by conducting thefollowing secondary assays. As previously described, D715-0284 is astereoisomer of molecule 768382-41-8.

1. HPLC deacylation assay. SIRT6 was incubated with compounds andmyristoyl-lysine peptide (corresponding to TNF-alpha sequence) at 37° C.The deacylation reactions were analyzed by reversed phasehigh-performance liquid chromatography on Kinetex C18 column bymonitoring the formation of the deacylated product at 214 nm. Theresults are shown in FIG. 1, which shows the area under the peakcorresponding to the deacylated product. Equally diluted DMSO served ascontrol.

2. Fluorometric deacetylation assay. SIRT6 was incubated with compoundsand fluorophore-labelled acetyl-lysine peptide (corresponding to histoneH3 sequence) at 37° C. FIG. 2 shows the effect of compounds on SIRT6deacetylation activity. Results are expressed as a percentage ofDMSO—treated control.

3. In vitro histone deacetylation assay. Chromatin was purified fromHEK293T cells by two steps of lysis (with Nonidet P-40 lysis buffer).SIRT6 was incubated with chromatin proteins and the reaction mixture isthen used for Western blot analysis. FIG. 3 shows deacetylation of H3K9on chromatin histones by SIRT6, in the presence or absence of compoundD715-0284. SIRT6 (0.5 μM) was incubated with chromatin fractionsisolated from HEK293T cells in the presence of 10 micromolar NAD+ andcompound D715-0284 or DMSO (control) at 37° C.

Example 4—Initial Pharmacophore Analysis

An initial pharmacophore analysis was performed with the BioviaDiscovery Studio package (DASSAULT SYSTEMES, USA) for three compounds,E677-1326, E677-0945 and D715-0284. As previously described, D715-0284is a stereoisomer of molecule 768382-41-8.

The pharmacophores were determined for the ligands, and may optionallybe generalized to describe a group of compounds as follows, includingfeature definitions which correspond to the data described below and inthe corresponding Figures.

Pharmacophore interpretation may optionally be described as follows,such that each compound may optionally be described as: a compoundhaving biological activity as an activator of SIRT6, which compoundbinds to SIRT6 as an activator, and having at least five chemicalfunctionalities for interacting with SIRT6, wherein said functionalitiesprovide a pharmacophoric motif selected from the group consisting of:

MOTIF 1 Charged Positive, Ring Aromatic, HBAcceptor, Charged Positive,Ring Aromatic

MOTIF 2 Charged Positive, HBAcceptor, Ring Aromatic, Ring Aromatic,HBAcceptor, HBAcceptor

MOTIF 3 Charged Positive, Hydrophobe Aromatic, HBAcceptor, RingAromatic, HBAcceptor

MOTIF 4 Hydrophobe, HBAcceptor, HBDonor, HBAcceptor, HBDonor

MOTIF 5 Hydrophobe, HBAcceptor, Ring Aromatic, HBDonor, HBDonor

wherein;

a HYDROPHOBE feature is defined as

-   -   a contiguous set of atoms that are not adjacent to any        concentrations of charge (charged atoms or electronegative        atoms), in a conformation such that the atoms have surface        accessibility;

a HYDROPHOBE AROMATIC feature is defined as

-   -   a contiguous set of atoms that are not adjacent to any        concentrations of charge (charged atoms or electronegative        atoms), in a conformation such that the atoms have surface        accessibility, arranged as an aromatic, including moieties        selected from the group consisting of neutral homocyclics,        heterocyclics, fused aromatics, polycyclics and substituted        aromatics;

a CHARGED POSITIVE feature is defined as

-   -   atoms or groups of atoms that are likely to be protonated at        physiological pH;

a RING AROMATIC feature is defined as

-   -   a contiguous set of atoms, in a ring conformation wherein the        atoms have surface accessibility, including moieties selected        from the group consisting of neutral homocyclics, heterocyclics,        fused aromatics, polycyclics and substituted aromatics;

a HBDONOR feature is defined as

-   -   a moiety that acts as a hydrogen bond donor;

and

a HBACCEPTOR feature is defined as

-   -   a moiety that acts as a hydrogen bond acceptor;

and wherein:

(a) a pharmacophore consisting of at least the following chemicalfeatures can be used to describe MOTIF 1:

Two Charged Positive features, two Ring Aromatic features and aHBAcceptor, in which each feature is represented by a sphere of 1.6Angstroms;

The positions of each feature are described as follows:

-   -   Charged Positive 1 has Cartesian XYZ co-ordinates of 2.473,        2.595, −0.526    -   Charged Positive 2 has co-ordinates of −2.782, 0.903, −0.968    -   Ring Aromatic 1 has co-ordinates of 3.692, 4.171, 1.434    -   Ring Aromatic 2 has co-ordinates of −1.784, −3.852, 1.181    -   HBAcceptor has co-ordinates of 2.468−1.358 0.921

and the distance and angle constraints for these are shown in FIG. 4A;

and wherein:

(b) a pharmacophore consisting of at least the following chemicalfeatures can be used to describe MOTIF 2:

A Charged Positive feature, three HBAcceptor features and two RingAromatic features, in which each feature is represented by a sphere of1.6 Angstroms;

The positions of each feature are described as follows:

-   -   Charged Positive has Cartesian XYZ co-ordinates of −3.757,        3.438, −0.387    -   HBAcceptor 1 has Cartesian XYZ co-ordinates of 1.508, 5.105,        −2.536    -   HBAcceptor 2 has Cartesian XYZ co-ordinates of −1.511, −1.486,        0.075    -   HBAcceptor 3 has Cartesian XYZ co-ordinates of 2.258, 0.942,        0.179    -   Ring Aromatic 1 has co-ordinates of 3.224, 3.61, −1.004    -   Ring Aromatic 2 has co-ordinates of 2.645, −5.558, 0.909

and the distance and angle constraints for these are shown in FIG. 4B;

and wherein:

(c) a pharmacophore consisting of at least the following chemicalfeatures can be used to describe MOTIF 3:

A Charged Positive feature, two HBAcceptor features, a HydrophobeAromatic feature and a Ring Aromatic feature, in which each feature isrepresented by a sphere of 1.6 Angstroms;

The positions of each feature are described as follows:

-   -   Charged Positive has Cartesian XYZ co-ordinates of −1.373, 1.99,        −1.36    -   HBAcceptor 1 has Cartesian XYZ co-ordinates of 2.714, −1.935,        1.105    -   HBAcceptor 2 has Cartesian XYZ co-ordinates of −0.84, −3.379,        0.729    -   Hydrophobe Aromatic has Cartesian XYZ co-ordinates of −3.134,        −4.648, 1.579    -   Ring Aromatic has Cartesian XYZ co-ordinates of 4.051, 3.593,        0.723

and the distance and angle constraints for these are shown in FIG. 4C;

and wherein

(d) a pharmacophore consisting of at least the following chemicalfeatures can be used to describe MOTIF 4:

A Hydrophobe feature, two HBAcceptor features, and two HBDonor features,in which each feature is represented by a sphere of 1.6 Angstroms;

The positions of each feature are described as follows:

-   -   Hydrophobe has Cartesian XYZ co-ordinates of 3.702, 4.163,        −1.853    -   HBAcceptor 1 has Cartesian XYZ co-ordinates of −0.406, 4.241,        −2.227    -   HBAcceptor 2 has Cartesian XYZ co-ordinates of −3.403, −3.629,        3.275    -   HBDonor 1 has Cartesian XYZ co-ordinates of −3.403, −3.629,        3.275    -   HBDonor 2 has Cartesian XYZ co-ordinates of −3.852, 0.454, 2.037

and the distance and angle constraints for these are shown in FIG. 4D;

and wherein

(e) a pharmacophore consisting of at least the following chemicalfeatures can be used to describe MOTIF 5:

A Hydrophobe feature, A HBAcceptor feature, a Ring Aromatic feature andtwo to HBDonor features, in which each feature is represented by asphere of 1.6 Angstroms;

The positions of each feature are described as follows:

-   -   Hydrophobe has Cartesian XYZ co-ordinates of 3.702, 4.163,        −1.853    -   HBAcceptor has Cartesian XYZ co-ordinates of −0.406, 4.241,        −2.227    -   Ring Aromatic has Cartesian XYZ co-ordinates of 1.288, 0.12,        −0.338    -   HBDonor 1 has Cartesian XYZ co-ordinates of −3.403, −3.629,        3.275    -   HBDonor 2 has Cartesian XYZ co-ordinates of −0.925, −4.787,        −0.207

and the distance and angle constraints for these are shown in FIG. 4E.

Preferably the compound induces SIRT6 catalyzed deacylation ordeacetylation.

The results are shown in FIGS. 4A-4E, each of which features thepharmacophore for the ligand on the left, with numbered features; andthe ligand on the right, with the corresponding parts of the ligandnumbered, to show correspondence to the numbered pharmacophore features.Colors shown are described in the tables below, to further aididentification.

A “centroid” is a feature that is determined by a surface model, such asof a ring aromatic (the ring aromatic vector is a “ring aromaticnormal”).

Hydrogen bond donors (HBDonor) and acceptors (HBAcceptors) are shownwith “tail” and “head” portions of the moiety labeled.

An “exclusion sphere” relates to the volume of space from which otherparts of the ligand are excluded. The “excluded volume” relates to areasexcluded by the ligand.

FIGS. 4A and 4B relate to exemplary pharmacophores for E677-1326.

For FIG. 4A, Table 4A shows a description of the type, coordinates andradius for each feature, plus color coordination information. Table 4Bshows the relative distance between each feature.

TABLE 4A # Type Color X Y Z Radius 1 Charged Positive 2.473 2.595 −0.5261.6 2 Ring_Aromatic_Centroid 3.692 4.171 1.434 1.6 2bRing_Aromatic_Normal 6.39728 3.36792 0.4159 1.6 3 HBAcceptor_Tail 2.468−1.358 0.921 1.6 3b HBAcceptor_Head 3.98 −1.827 3.469 2.2 4Charged_Positive −2.782 0.903 −0.968 1.6 5 Ring_Aromatic_Centroid −1.784−3.852 1.181 1.6 5b Ring_Aromatic_Normal −0.4093 −1.1955 0.95005 1.6

TABLE 4B Feature Distance # 2 3 4 5 1 2.706 4.21 55.38 7.912 2 5.6867.64 9.717 3 6.02 4.936 4 5.313

For FIG. 4B, Table 5A shows a description of the type, coordinates andradius for each feature, plus color coordination information. Table 5Bshows the relative distances between each feature.

TABLE 5A # Type Color X Y Z Radius 1 Charged Positive −3.757 3.438 0.3871.6 2 HBAcceptor_Tail 1.508 5.105 −2.536 1.6 2b HBAcceptor_Head −0.3826.736 −4.199 2.2 3 Ring_Aromatic_Centroid 3.224 3.61 −1.004 1.6 3bRing_Aromatic_Normal 2.23718 5.03675 1.44356 1.6 4Ring_Aromatic_Centroid 2.645 −5.558 0.909 1.6 4b Ring_Aromatic_Normal3.08508 −5.558 3.87655 1.6 5 HBAcceptor_Tail −1.511 −1.486 0.075 1.6 5bHBAcceptor_Head −3.864 −3.322 −0.232 2.2 6 HBAcceptor_Tail 2.258 0.9420.179 1.6 6b HBAcceptor_Head 3.358 −1.195 1.975 2.2

TABLE 5B Feature Distance # 2 3 4 5 6 1 5.926 7.01 11.117 5.432 6.537 22.743 11.263 7.705 5.026 3 9.383 7.646 4.286 4 5.878 6.552 5 4.485

FIG. 4C shows an exemplary pharmacophore for E677-0945. For FIG. 4C,Table 6A shows a description of the type, coordinates and radius foreach feature, plus color coordination information. Table 6B shows therelative distances between each feature.

TABLE 6A # Type Color X Y Z Radius 1 Charged_Positive −1.373 1.99 −1.361.6 2 Hydrophobe_Aromatic −3.134 −4.648 1.579 1.6 3 HBAcceptor_Tail2.714 −1.935 1.105 1.6 3b HBAcceptor_Head 4.618 −2.18 3.41 2.2 4Ring_Atomatic_Centroid 4.051 3.593 0.723 1.6 4b Ring_Aromatic_Normal1.93713 4.39995 2.69287 1.6 5 HBAcceptor_Tail −0.84 −3.379 0.729 1.6 5bHBAcceptor-Head −1.161 −0.516 −0.108 2.2

TABLE 6B Feature Distance # 2 3 4 5 1 7.470 6.179 6.027 5.786 2 6.46410.967 2.756 3 5.700 3.855 4 8.516

FIGS. 4D and 4E show exemplary pharmacophores for D715-0284. For FIG.4D, Table 7A shows a description of the type, coordinates and radius foreach feature, plus color coordination information. Table 7B shows therelative distances between each feature.

TABLE 7A # Type Color X Y Z Radius 1 Hydrophobe 3.702 4.163 −1.853 1.6 2HBAcceptor_Tail −0.406 4.241 −2.227 1.6 2b HBAcceptor_Head −2.138 6.445−3.297 2.2 3 HBDonor_Tail −3.403 −3.629 3.275 1.6 3b HBDonor_Head −0.446−3.31 3.671 2.2 4 HBAcceptor_Tail −3.403 −3.629 3.275 1.6 4bHBAcceptor_Head −5.373 −4.381 5.409 2.2 5 HBDonor_Tail −3.852 0.4542.037 1.6 5b HBDonor_Head −5.458 0.707 −0.484 2.2 (6) ExclusiveSphere−4.799 −3.511 1.826 1.2 (7) ExcludedVolume_1.11 −1.194 −0.064 −0.486 1.2(8) ExcludedVolume_1.12 2.653 −1.743 0.59 1.2

TABLE 7B Feature Distance # 2 3 4 5 1 4.126 11.726 11.726 9.271 2 10.05910.059 6.663 3 — 4.290 4 4.290

For FIG. 4E, Table 8A shows a description of the type, coordinates andradius for each feature, plus color coordination information. Table 8Bshows the relative distances between each feature.

TABLE 8A # Type Color X Y Z Radius 1 Hydrophobe 3.702 4.163 −1.853 1.6 2HBAcceptor_Tail −0.406 4.241 −2.227 1.6 2b HBAcceptor_Head −2.138 6.445−3.297 2.2 3 Ring_Aromatic_Centroid 1.288 0.12 −0.338 1.6 3bRing_Aromatic_Normal 1.03402 1.29691 2.40979 1.6 4 HBDonor_Tail −3.403−3.629 3.275 1.6 4b HBDonor_Head −0.446 −3.31 3.671 2.2 5 HBDonor_Tail−0.925 −4.787 −0.207 1.6 5b HBDonor_Head 2.06 −4.549 −0.025 2.2 (6)ExclusiveSphere −1.194 −0.064 −0.486 1.2 (7) ExcludedVolume_1.11 2.653−1.743 0.59 1.2 (8) ExcludedVolume_1.12 −4.799 −3.511 1.826 1.2

TABLE 8B Feature Distance # 2 3 4 5 1 4.126 4.947 11.726 10.209 2 4.83910.059 9.266 3 7.008 5.385 4 4.428

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

What is claimed is:
 1. A method of a treatment with a SIRT6 activator,for a subject in need of treatment thereof, comprising administering acompound selected from the group consisting of: A

B

7

10

11

12

15

16

20

21

22

24

27

28

29

30

35

36

39

50

51

52

55

56

57

58

63

64

to the subject.
 2. The method of claim 1, wherein the compound isselected from the group consisting of A and B.
 3. The method of claim 2,wherein the subject is suffering from a condition amenable to treatmentwith the SIRT6 activator and wherein the condition is selected from thegroup consisting of obesity, an obesity-related disease, and afat-related metabolic disorder.
 4. The method of claim 2, wherein thesubject is suffering from a condition selected from the group consistingof lipid disorders including hyperlipidemia and hyperuricemia, insulinresistance, noninsulin dependent diabetes mellitus (NIDDM, or Type IIdiabetes), insulin dependent diabetes mellitus (IDDM or Type Idiabetes), diabetes-related complications including microangiopathiclesions, ocular lesions, retinopathy, neuropathy, and renal lesions,cardiovascular disease (including cardiac insufficiency, coronaryinsufficiency, and high blood pressure), atherosclerosis, atheromatousdisease, stroke, hypertension, Syndrome X, gallbladder disease,osteoarthritis, sleep apnea, uterine cancer, breast cancer, colorectalcancer, kidney cancer, and gallbladder cancer, high cholesterol levels,complications of pregnancy, menstrual irregularities, hirsutism,muscular dystrophy, infertility, a weight-related disorder and increasedsurgical risk.
 5. The method of claim 2, wherein the subject issuffering from a condition amenable to treatment with the SIRT6activator and wherein the subject has a body fat percentage of at leastat 20%.
 6. The method of claim 2, wherein the subject is suffering froma condition amenable to treatment with the SIRT6 activator and whereinthe condition is selected from the group consisting of type 2 diabetes,prediabetes, glucose intolerance, insulin insensitivity, hyperglycemia,hypoglycemia, gestational diabetes, drug-induced diabetes; overweight,obesity, high percent body fat and/or body composition and/or body fatlocation, dyslipidemia defined as abnormal levels of blood lipids; andnon-alcoholic fatty liver disease (NAFLD).
 7. The method of claim 2,wherein the subject is suffering from a condition amenable to treatmentwith the SIRT6 activator and wherein the condition is selected from thegroup consisting of cardiovascular disease, neurodegenerative disease,premature aging syndrome and aging.
 8. The method of claim 2, whereinthe subject is suffering from a condition amenable to treatment with theSIRT6 activator and wherein the condition is selected from the groupconsisting of breast cancer, cervical cancer, ovary cancer, endometrialcancer, melanoma, uveal melanoma, bladder cancer, lung cancer,pancreatic cancer, colorectal cancer, prostate cancer, leukemia, acutelymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma,Burkitt's lymphoma, multiple myeloma, Non-Hodgkin's lymphoma, myeloidleukemia, acute myelogenous leukemia (AML), chronic myelogenousleukemia, thyroid cancer, thyroid follicular cancer, myelodysplasticsyndrome (MDS), fibrosarcomas and rhabdomyosarcomas, teratocarcinoma,neuroblastoma, glioma, glioblastoma, benign tumor of the skin,keratoacanthomas, renal cancer, anaplastic large-cell lymphoma,esophageal cancer, follicular dendritic cell carcinoma, seminal vesicletumor, epidermal carcinoma, spleen cancer, bladder cancer, head and neckcancer, stomach cancer, liver cancer, bone cancer, brain cancer, cancerof the retina, biliary cancer, small bowel cancer, salivary glandcancer, cancer of uterus, cancer of testicles, cancer of connectivetissue, myelodysplasia, Waldenstrom's macroglobinaemia, nasopharyngeal,neuroendocrine cancer, mesothelioma, angiosarcoma, Kaposi's sarcoma,carcinoid, fallopian tube cancer, peritoneal cancer, papillary serousmtillerian cancer, malignant ascites, gastrointestinal stromal tumor(GIST), Li-Fraumeni syndrome, and Von Rippel-Lindau syndrome (VHL).